Tyrosyl-tRNA synthetase has a noncanonical function in actin bundling

Dominant mutations in tyrosyl-tRNA synthetase (YARS1) and six other tRNA ligases cause Charcot-Marie-Tooth peripheral neuropathy (CMT). Loss of aminoacylation is not required for their pathogenicity, suggesting a gain-of-function disease mechanism. By an unbiased genetic screen in Drosophila, we link YARS1 dysfunction to actin cytoskeleton organization. Biochemical studies uncover yet unknown actin-bundling property of YARS1 to be enhanced by a CMT mutation, leading to actin disorganization in the Drosophila nervous system, human SH-SY5Y neuroblastoma cells, and patient-derived fibroblasts. Genetic modulation of F-actin organization improves hallmark electrophysiological and morphological features in neurons of flies expressing CMT-causing YARS1 mutations. Similar beneficial effects are observed in flies expressing a neuropathy-causing glycyl-tRNA synthetase. Hence, in this work, we show that YARS1 is an evolutionary-conserved F-actin organizer which links the actin cytoskeleton to tRNA-synthetase-induced neurodegeneration.


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March 2021

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Structured illumination microscopy of larval NMJs was performed on a Zeiss ELYRA S.1 microscope equipped with a 63x Plan-Apochromat objective (1.4 NA). Act5C::GFP signal at the larval NMJs was acquired using a Nikon Ni-E upright microscope equipped with a Yokogawa CSU-W1 spinning-disk head, an Andor iXon 897U EMCCD camera and Nikon Elements AR software. A 60x (NA 1.4) oil immersion objective. Trypsin digested peptides from the FLAG-YARS1 immunoprecipitation were analyzed using a Q Exactive Orbitrap mass spectrometer (Thermo Fisher). Advancing versions of ImageJ from 1.45a (6 February 2011) including the latest 1.53t (24 August 2022) were used in the study (ImageJ (nih.gov)). The EP-targeted genes' annotations, expression data and functional annotations were retrieved from advancing versions of FlyBase, including the latest FB2022_06 (FlyBase Homepage). We used the PANTHER Classification System, version 16.0 to retrieve gene ontology (GO) information (pantherdb.org). The quantitative proteomics software package MaxQuant version 1.5.3.8 was used to obtain label-free quantification (LFQ) from the mass-spec data (MaxQuant). ClustalOmega Version 1.2.2 was used for protein sequence alignment (Clustal Omega < Multiple Sequence Alignment < EMBL-EBI). DroID data base version DroID_v2018_08 (DroID: The Comprehensive Drosophila Interactions Database (droidb.org)).
A separate "Data availability" section is provided in the manuscript. All data generated and/or analysed during this study are included in this article (and its supplementary files). The source data underlying Figures 2, 3, 4, 5 and 6, and Supplementary Figures 1, 2 , 4, 5, 7, 8, 9, and 10 are provided as a Source Data file. The mass spectrometry data are deposited at the PRIDE database (PXD037630). All other relevant data (e.g. ImageJ macro scripts) are available from the corresponding author on reasonable request. The human biomaterials are available subject to MTA. Databases and softwares relevant to the study include (ImageJ (nih.gov)); FlyBase, (FlyBase Homepage); PANTHER Classification System (pantherdb.org); the quantitative proteomics software package MaxQuant (MaxQuant); ClustalOmega (Clustal Omega < Multiple Sequence Alignment < EMBL-EBI); DroID data base DroID: The Comprehensive Drosophila Interactions Database (droidb.org)).
A separate "Data availability" section is provided in the manuscript. All data generated and/or analysed during this study are included in this article (and its supplementary files). The source data underlying Figures 2, 3, 4, 5 and 6, and Supplementary Figures 1, 2 , 4, 5, 7, 8, 9, and 10 are provided as a Source Data file. The mass spectrometry data are deposited at the PRIDE database (PXD037630). All other relevant data are available from the corresponding author on reasonable request. The human biomaterials are available subject to MTA. Databases and softwares relevant to the study include (ImageJ (nih.gov)); FlyBase, (FlyBase Homepage); PANTHER Classification System (pantherdb.org); the quantitative proteomics software package MaxQuant (MaxQuant); ClustalOmega (Clustal Omega < Multiple Sequence Alignment < EMBL-EBI); DroID data base DroID: The Comprehensive Drosophila Interactions Database (droidb.org)). A separate "Code availability" section is provided in the manuscript as well, stating that "Relevant code and classifiers (e.g. ImageJ macro scipts) are available from the corresponding author on reasonable request".
The findings apply for both sexes. No individuals level information was reported.
Dermal fibroblasts from one YARS1-E196K/WT patient (male, 44 y) and one control individual (male, 42 y) were sampled using standard procedures and after obtaining their written informed consent. The study complies with the ethical guidelines of the Medical University-Sofia, Bulgaria and University of Antwerp, Belgium and was approved by the respective local institutional review boards.
The CMT patient was recruited upon clinical examination by Dr. Ivaylo Tournev who is an expert neurologist at the Medical University-Sofia, Bulgaria. Dr. Tournev is included as a co-author in this manuscript. The subtype of CMT caused by the dominant YARS1-E196K mutation is rare and limited to a single large pedigree from Bulgaria. This might present a bias however we do not have evidence to either prove of disprove this at the moment.
We have complied with all relevant ethical regulations for animal and human testing and research. The study was approved by the Ethical committee of the University of Antwerp and University Hospital Antwerp (14/15/188). Informed consent was obtained from all individuals included in the study.

March 2021
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All studies must disclose on these points even when the disclosure is negative.  From the FRAP experiment we excluded boutons with poor curve fitting parameter (R² < 0.95). From cell migration experiment in SH-SY5Y cells low-quality wounds (irregular, occurrence of cell debris) were removed (<10% of wells).
In the retinal degeneration screen at least 20 female flies from the control and test genotypes were compared with each other. Genetic crosses for positive hits were repeated at least three independent times. Other Drosophila-related experiments also were performed on larvae or adults coming from at least three independent genetic crosses. The in vitro pelleting assays were performed at least three independent times, except for HARS1 (two times). TIRF experiments were performed two independent times at two different concentrations. Each figure contains information in the legend on the independent experiments performed. All attempts at replication were successful. As we describe in the Methods section, the TIRF experiments were repeated several times and in each case F-actin bundle and cable formation was induced by YARS1; however, there was frequent "stickiness" observed in the presence of YARS1 that prevented robust scoring of real-time events with TIRF.
In the experiments involving Drosophila, experimental groups were formed by choosing animals with the correct genotype (usually controlled by the absence or presence of genetic markers), at the appropriate developmental stage (adult or third instar wandering larvae), and gender (females). In the cellular experiments and TIRF, imaging was randomized to different parts of the slide.
Many of our studies involved unbiased way of quantification of the phenomena thus we did use blinding.